Agent for the removal of turbidity in biological samples

ABSTRACT

Agent for the removal of turbidity in biological samples comprising 0.5-10 mM Phenol, 0.5-15% polyoxyethylated triglyceride and at least one non-ionic ten-side in a range of 0.5-15% capable in dissolving the polyoxyethiylated triglyceride

[0001] The invention relates to an agent for the removal of turbidity inbiological samples, especially in serum or plasma samples.

[0002] Turbidity typically occurs in plasma or serum samples (lipemicsamples) which have an increased content of triglyceride-richlipoprotein particles, e.g. chylomicrons.

[0003] Known agents for removal of turbidity are especially necessaryfor photometric analysis of lipemic samples in chemical or clinicaldiagnosis. If the component to be analysed absorbs at a wavelengthcorresponding to the absorbing wavelength of the tiiglycerides causingthe turbidity, correct photometric analysis of the component in questionwill be difficult if not impossible. This applies especially when theconcentration of the component to be determined is very low, e.g. in thecase of the analyte Cell Reactive Protein (CRP).

[0004] Therefor a typical example for a photometric procedure in whichinterference with turbidity must be excluded is the assay of CRP. Theassay uses anti-human CRP antibodies which specifically react with theCRP in the sample to form insoluble aggregates. The assay is initiatedby the addition of the antibodies and the increase inCRP-antibody-aggregates is measured photometrically at 340 nm. Thetiiglycerides in lipemic samples also absorb at this wavelength so thatthere is the risk of overlapping extinction signals.

[0005] To resolve this problem with lipemic samples several agents areknown in the prior art which can be added in order to remove turbidity.In this connection U.S. Pat. No. 4,708,939 discloses an agent comprisingpolyethoxylated triglyceride and a secondary n-alkane sulphonate inaqueous solution. The known agent requires relatively high detergentconcentrations which can affect certain assays, especiallyimmunoturbidimetric assays. In addition n-alkane sulphonate is anagressive detergent which again can inhibit the reaction of interest.The known agent therefor cannot give total clearing and optimumantibody-antigen reaction.

[0006] The object of the present invention is to provide an agent forthe removal of turbidity which is especially adapted to CRP assays, butcan be used also for other assays.

[0007] An agent according to the invention comprises 0.5 to 10 mMphenol, 0.5 to 15% of an polyoxyethylated triglyceride and 0.5 to 15% ofat least one further non-ionic tenside. Preferably a combination of twofurther non-ionic tensides is included.

[0008] In the agent according to the invention the polyethoxylatedtriglyceride (Triglyceridethoxylate) is necessary to obtain totalclearing of the sample. A suited polyethoxylated triglyceride can have aHLB value in the range of 4-16, preferably 61 The preferred range ofconcentration is 0.5 to 2%. Preferred polyoxyethylated triglycerides areavailable under the tradenames Mulsifan RT 163(Zschimmer & Schwarz GmbH& Co.) or Tagat CH-40 (Goldschmidt AG).

[0009] The polyoxyethylated triglycerides alone cannot dissolve in themixture resulting in a precipitate. Dissolution requires at least onefurther non-ionic tenside that assits in the solubilisation of thepolyethoxylated triglycerides in the sample.

[0010] Typical examples of non-ionic tensides which can be used include:Thesit, Tergitol, Triton, Brij, Nonidet P-40, Tween 20 (Sigma-AldrichLtd.).

[0011] The required non-ionic tensides are straight or branchedpolyoxyethylene ethers with a low degree of oxyethylation (2-10oxyethylene units per molecule) with 10-18 carbon atoms.

[0012] Preferably the further non-ionic tenside is polyoxyethylene (8)isotridecylether which is available under the tradename Genapol X 080.This non-ionic tenside dissolves in the reaction mixture but alone doesnot have a clearing effect. It is required to solubilise thepolyethoxylated triglycerides (as stated above). A preferred range ofconcentration in which the non-ionic tenside can be used is 0.5 to 2%.

[0013] A further preferred non-ionic tenside ispolyoxyethylene-10-tridecylether which can be purchased from Sigma. Alsothis tenside dissolves in the reaction mixture and assists thesolubilisation of the polyethoxylated triglycerides. A preferred rangeof concentration is 0.5 to 2%.

[0014] In a preferred embodiment a combination of polyoxyethylene (8)isotridecylether and polyoxyethylene-10-tridecylether is provided. Suchcombination allows a very effective dissolution of the polyoxyethylatedtriglycerides and thereby improves the clearing action of the agentaccording to the invention.

[0015] Phenol finally has no dissolving effect on the lipids but actssynergistically with the tensides to increase their clearing action andremove turbidity. The presence of phenol helps to reduce the effectiveconcentrations of the tensides needed. In the absence of phenol highertenside concentrations would be necessary which would lead to sampleshaving a too high viscosity.

[0016] The components in the agent according to the invention are knownsubstances which already are used in agents for the removal of turbidityknown in the prior art.

[0017] The use of phenol as synergistically acting component is knownfrom EP 0004857. From EP 0004857 it is also known to use Genapol asnon-ionic tenside. The use of further non-ionic tensides is known fromthe U.S. Pat. No. 4,708,939 cited above. The prior art, however,discloses only the use of the single components or sub-combinationsthereof. None of them shows the combination of all of them as in theagent according to the invention,

[0018] It surprisingly turned out that all components included in theagent according to the invention are necessary to effectively removeturbidity with minimal effects to a possible antibody-antigen reaction.Theoretically Genapol and polyoxyethylene-10-tridecylether togethercould effect clearing. However, the concentrations required for clearingare so high that any antibody-antigen reaction is totally inhibited. Ifaccording to the invention Mulsifan and Phenol are added theconcentrations of Genapol and polyoxyethylene-10-tridecylether can bereduced which allows clearing without affecting immunological reactions.

[0019] In principle the agent according to the invention can be used inall immunoassay formats where there is a likelihood of interaction ofcontributing to lipemia.

[0020] The example given below uses an immunoturbidimetric format. Thoseskilled in the alt would be aware of associated benefits for otherformats including (but not limited to) latex-enhanced assays, magneticparticle chemiluminescent immunoassays, immunofluorescent assays(polarised and non-polarised), ELISA's, immunocluomatographic assays, orany assay format requiring reduction of lipemic and/or other associatednon-specific binding problems where the benificial maintenance ofantibody binding characteristics is mediated by the combination ofreactants in the present invention.

[0021] In the following a typical formulation of a clearing agentaccording to the invention to be used in an assay for the detection ofCRP is described. Furthermore an example is added showing the removal ofturbidity effected by agent according to the invention in lipemicsamples.

[0022] Formulation of a Clearing Buffer (R1) for a CRP-Assay: ReagentRange Function Purified water Tris (100 mM)/Liter  50-300 mM Buffer NaCl(100 mM)/Liter  50-150 mM/ Assists clarification & reaction. PEG 6000(2%)(w/w)   1-3% Req. for optimisation of reaction rate Phenol (2Mm)(Liter) 0.5-10 mM Lipid clearing component Polyoxyethylene 10 0.5-2%Lipid clearing component tridecylether (1.0%)(v/v) Genapol X 80(1.0%)(v/v) 0.5-2% Lipid clearing component Mulsifan RT 163 (1.1%)(v/v)0.5-2% Lipid clearing component NaN₃ Anti microbial agent 4 M HCl pHreagent to 7.5 Gentamicin sulphate soln. Stabilizer

[0023] The buffer R1 is adjusted to pH 7.5 (possible range pH 3-9) andis preferably used at a temperature of 37° C. (possible range 15-40°C.).

[0024] For the CRP-assay 250 μl of R1 are mixed with 18 μl of thesample. Then 30 μl of an antiserum solution (R2; includes anti CRPantibodies) are added. After mixing of the sample with R1 buffer and R2antiserum solution, the CRP in the sample reacts specifically with theanti-human CRP antibodies of R2 to yield insoluble aggregates. Theabsorbance of these aggregates is proportional to the CRP concentrationin the sample.

[0025] The purpose of the R1 buffer is to dissolve lipids in lipemicsamples and to provide optimum conditions for the immunogical reaction.

[0026] Removal of Turbidity in Lipemic Samples

[0027] In this test a clearing buffer (R3) was used with the followingformulation: 100 mM Tris buffer adjusted to pH 7.5 with HCl, 100 MmNaCl, 2% Polyoxyethylene glycol 6000, 2 mM Phenol, 1.0% Polyoxyethylene10 tridecylether, 1.0% Genapol X 80 and 1.0% Mulsifan RT 163.

[0028] In a cuvette 18 μl of strongly lipemic serum were mixed at 37° C.with 250 μl of R3. The change of absorbance at 340 nm was determinedagainst dependence on time. The results are illustrated graphically inFIG. 1.

[0029] The course of the change of absorbance illustrated in FIG. 1shows that in the case of a sample/reagent ratio of 18 ul: 250 ul aturbidity of about 1.5 is reduced within about 1.5 minutes to a clearedlevel of about absorbance 0.1 in comparison with the reagent blank.Therefore, after 1.5 minutes complete removal of turbidity has beenachieved.

1. Agent for the removal of turbidity, in biological samples comprising 0.5 10 mM Phenol 0.5 15% polyoxyethylated triglyceride and at least one non-ionic tenside in a range of 0.5-15% capable in dissolving the polyoxyethylated triglyceride.
 2. Agent according to claim 1, characterised in that the non-ionic teniside is selected from the group including: polyoxyethlene-10-tridecylether and poyloxyethylene (8) isotridecylether.
 3. Agent according to claim 2, characterised in that the agent comprises a combination of polyoxyethlene-10-tridecylether and poyloxyethylene (8) isotridecylether.
 4. Agent according to claims 1 to 3, characterised in that the concentration of the non-ionic tenside is 0.5-2%.
 5. Agent according to claim 1, characterised in that the concentration of the polyoxyethylated triglyceride is 0.5-2%.
 6. Agent according to one of the claims 1-4, characterised in that it is used in assays to detect CRP. 